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Abstract

A newly described cytokine, interleukin-27 (IL-27), that activates naive CD4 T cells, has recently been shown to be an anti-HIV cytokine. However, the effect of HIV infection on IL-27 expression has not been characterized. We found that clinical characteristics, including HIV viral load, hepatitis C virus coinfection, and CD4 T cell counts, were associated with changes in serum IL-27. Overall, our results suggest circulating HIV may suppress IL-27, a critical concept in treatment development with this cytokine.

HIV infection causes dysregulated cytokine production [1–5], resulting in impaired immunity characteristic of HIV/AIDS. Interleukin-27 (IL-27) is a newly described IL-12 family cytokine critical in development of Th1 responses [6–8]. IL-27 also regulates inflammatory responses in monocytes/macrophages [9,10] and CD4 T cells [6,11], which are targets of HIV. The effect of HIV on IL-27 has not been investigated. Interestingly, studies have demonstrated that IL-27 can inhibit HIV replication [12–14], leading to the possibility of IL-27 administration as anti-HIV therapy. This study characterizes for the first time how HIV affects IL-27 expression.

In accordance with Queen's University Research Ethics Board, informed consent was obtained from HIV-positive (n = 32) and HIV-negative (n = 11) individuals. Group 1 (n = 6) was naive to highly active antiretroviral therapy (HAART) with a median viral load of 24 927 copies/ml, group 2 (n = 10) was receiving HAART with low viral load (<500 copies/ml), group 3 (n = 9) was receiving HAART with low viral load and coinfected with hepatitis C virus (HCV), and group 4 (n = 7) was receiving HAART with high viral load (>500 copies/ml, median of 3003 copies/ml). Of the group 4 patients, three were tolerant/nonadherent to HAART, two were two-class resistant [nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors], and one was one-class resistant (NRTI). Ten patients (n = 2, 4, 3, and 1 in groups 1, 2, 3, and 4, respectively) had samples drawn several months apart and, therefore, are included twice to enhance statistical power. CD4 T cell counts and viral loads were obtained during routine clinic visits (Clinical Immunology Outpatient Clinic, Hotel Dieu Hospital, Kingston, Ontario, Canada). Due to the small sample size and nonnormal distribution of key variables, nonparametric tests (Spearman's correlations, Mann–Whitney U-test, Kruskal–Wallis test) were used for analysis, using SPSS, version 17.0 for windows (SPSS Inc., Chicago, Illinois, USA). A P value less than 0.15 was considered a trend of clinical relevance, whereas a P value less than 0.05 was used to define statistical significance.

We observed an overall moderate negative correlation between IL-27 and viral load using Spearman's ρ (ρ = −0.201, P = 0.149; Fig. 1b, top). Results were similar (ρ = −0.299) in group 1 (Fig. 1b, middle), but the drop in sample size (from 53 to 8) resulted in a substantial loss of statistical power (P = 0.47). Interestingly, in group 4 (Fig. 1b, bottom), no association was found. Groups 2 and 3 present no detectable viral load and, therefore, were not included. When HIV-infected patients were grouped as either naive to or on HAART (Fig. 1c), the Mann–Whitney test found no difference in the mean rank of IL-27 between the two groups, indicating no association of HAART and HIV viral load.

We investigated whether HCV coinfection might influence IL-27 and observed a decrease in IL-27 in patients coinfected with HCV compared to those monoinfected with HIV (Fig. 1d), which fell just short of statistical significance (P = 0.093). Additionally, we employed the Kruskal–Wallis test among all study participants (overall) and for each of the HIV-infected groups to examine whether IL-27 expression differed with CD4 T cell count (Fig. 1e). The overall group indicated a noteworthy trend of IL-27 expression within CD4 groups (P = 0.153); IL-27 peaked within the moderate CD4 T cell count group (200–350) and decreased in the low CD4 T cell count group (<200). This was a consistent trend of IL-27 within CD4 groups, with moderate CD4 T cell counts (200–350) showing highest IL-27 in four out of five groups.

Recent studies identify IL-27 as an anti-HIV cytokine [12–14], indicating a potential role for IL-27 in the control of HIV replication. Herein, we describe for the first time how IL-27 levels are modulated in HIV infection. We observed a negative correlation between viral load and IL-27; high viral load may suppress IL-27, a pathogenic mechanism used by HIV to downregulate immune responses. This is supported by studies showing low expression of the related cytokine, IL-12, in HIV infection [15,16]. The negative correlation also substantiates previous findings showing IL-27-mediated inhibition of viral replication [12–14]. The loss of the negative correlation in group 4 could be attributed to widespread loss of normal immunologic function and/or the development of tolerance/resistance to HAART. Whether suppression of IL-27 is mediated via host response or directly by HIV remains to be investigated.

We observed no significant difference in IL-27 among patients naive to HAART versus those receiving HAART, consistent with a recent study showing no effect of HAART on the IL-27-related cytokine, IL-12, and its overproduction observed in acute infection [1]. Our finding that HCV coinfection was associated with a significant decrease in IL-27 indicates that HCV may suppress IL-27, paralleling previous studies showing HCV core protein inhibits IL-12 [17–19]. On the contrary, studies have shown enhanced circulating cytokines in HCV infection [20,21], including IL-12, whereby HCV genotype 1 was associated with a significant increase in IL-12 expression [20]. Further work investigating HCV viral loads and genotypes in coinfected participants might provide insight into IL-27 suppression.

We observed CD4 T cell count categories to significantly differ in IL-27 expression. In four out of five groups, a boost in IL-27 from high CD4 T cell counts (>350) to moderate (200–350), followed by a decline in IL-27 at low CD4 cell counts (<200) was observed. We hypothesize the initial boost in IL-27 to indicate host response to viral insult. IL-27 can inhibit HIV replication [12–14,21] and, therefore, upregulating IL-27 could be a protective response to decreasing CD4 T cells and increasing viral load. As disease weakens immunity, the virus may then suppress IL-27.

Our findings indicate HIV viral load, HCV coinfection, and CD4 T cell counts are strongly associated with changes in serum IL-27. This study is the first to report how HIV infection influences IL-27 expression. Understanding how HIV affects IL-27 is a critical step in determining the potential of IL-27 as a therapeutic adjunct to HIV treatments.

Acknowledgements

The authors would like to thank blood donors for their participation in this study, along with Jenna Ekborn and Catherine Fuller (Clinical Immunology Outpatient Clinic, Hotel Dieu Hospital, Kingston, ON) for their assistance in participant recruitment. This work was supported by a Research Initiation Grant from Queen's University and the Canadian Foundation for Innovation.

C.G. processed serum samples, performed ELISAs, and contributed to concept and study design. W.M.H. performed all statistical analysis and assisted with interpretation of results. N.F.C.M. assisted with the processing of serum samples. W.W. is the director of the immunology clinic at Hotel Dieu Hospital and oversaw sample collection as well as assisted with concept and study design. K.G. is the corresponding author.

C.G. is supported by a studentship from the Ontario HIV Treatment Network.

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